This invention relates to probes for detecting chondrogenesis.
Cartilage is a dense connective tissue that comprises part of the skeleton in adult humans. Cartilage provides support and attachment points for body structures, protects underlying tissues, and provides structural models in which many bones develop.
Cartilage is largely comprised of cells, called chondrocytes, embedded in an extracellular matrix. The extracellular matrix mainly consists of collagen type II and proteoglycans, the components of which are exuded into the intercellular space by the chondrocytes, where they are assembled to form macromolecules. The chondrocytes make up about 5% of the volume of the cartilage tissue of an adult individual.
Chondrocytes are formed by differentiation of mesenchymal progenitor cells (MPCs), also called mesenchymal stem cells (MSCs). These cells form chondrocytes during embryonic development (see below). In addition, MPCs are found in many tissues of the adult body, and are multipotent in that they can differentiate into a number of different cell types (see FIG. 1). The process of differentiation from progenitor or stem cell to chondrocyte is called chondrogenesis.
Chondrogenesis in Development and Growth
Chondrocytes are an essential component to bone development and growth, a process called endochondral ossification. For example, most bones of the human skeleton develop from masses of hyaline cartilage. This cartilage is formed by chondrocytes that are differentiated from MPCs. The first part of chondrogenesis is the formation of a precartilage condensation, where the MPCs come together through cell-cell interactions and there is some proliferation. After condensation, the progression phase of chondrogenesis occurs, with cells beginning to produce molecules characteristic of cartilage, such as type II collagen. There is expansion of the cartilaginous tissue by production of large amounts of extracellular matrix containing mainly collagen and proteoglycans. This leads to the formation of a cartilage anlagen for a given bone.
In certain regions of this cartilage, the chondrocytes further differentiate into hypertrophic chondrocytes that secrete bone-related molecules and their surrounding extracellular matrix is calcified. The cells then die by apoptosis (programmed cell death). Blood vessels invade the calcified cartilage and osteoblasts (the cells responsible for bone formation) are attracted. Chondroclastic cells remove the cartilage and the osteoblasts lay down new bone. This bone is later remodeled by osteoclasts and osteoblasts to form mature bone.
Other regions of the cartilage anlagen are not removed during development; specifically the growth plates and the articular cartilages. Growth plate chondrocytes enter a programmed pathway in which they proliferate for some time and then become hypertrophic and die, with replacement of the hypertrophic region by the mechanism described above, involving blood vessel invasion, etc. This mechanism allows bones to grow longitudinally until puberty. Articular cartilage resembles the growth plate in the neonatal stage, but eventually there is formation of a cartilage in which the chondrocytes no longer proliferate or hypertrophyxe2x80x94a permanent cartilage. Articular cartilage is responsible for weight-bearing and shock absorption in joints. It is the cartilage that breaks down in degenerative arthritic diseases. Other permanent cartilages include those that form rings in the trachea or the cartilage of the nose and ears.
Because it is important, in certain instances, to monitor progression of chondrogenesis in both formation of cartilage and bone, there is a need for markers and probes to detect and ascertain the extent of these processes.
Chondrogenesis in Natural Repair and Regeneration
In addition to formation of bone during development, chondrocytes are also involved in the formation of bone in repair of bone fractures. Within a typical fracture site, there is both intramembranous bone formation (where there is no cartilage intermediate) and endochondral bone formation (in which cartilage is first formed and then replaced by bone, in a manner similar to that seen in development).
Immediately after a fracture, a fibrous clot is formed and granulation tissue results as macrophages and other cells invade it. This is called the xe2x80x9cexternal callus.xe2x80x9d Bone begins to be made by osteoblasts adjacent to the fracture, forming a hard callus. At the same time, progenitor cells from the surrounding tissues proliferate and begin to differentiate into chondrocytes within the granulation tissue. This cartilaginous callus is later replaced by bone tissue, similar to the process in which hyaline cartilage of a developing bone is replaced.
Because the endochondral component is important to effect proper bone repair, there is a need for markers and probes to detect and ascertain the extent of chondrogenesis in this process. This is especially true when a so-called non-union occurs, in which the fracture does not heal. Understanding what stage the repair has reached would aid in the choice of the remedial treatment.
Chondrogenesis in Therapeutic Cartilage Repair and Regeneration
In a separate application, the use of implanted mesenchymal progenitor cells to produce repair of cartilaginous tissues would benefit from knowledge of the stage of differentiation that the cells have reached. For example, in articular cartilage repair, the undifferentiated progenitor cells, either injected or implanted into area of the body where there is defective cartilage, is a possible treatment modality. The course of such treatment includes the sampling (biopsy) of repair tissue at some time after implantation. In order to aid in decisions regarding the treatment, it is important to know the stage of chondrogenic differentiation that the implanted cells have reached. Thus, there is a need for markers and probes of chondrogenesis to detect and ascertain the extent of this process.
Chondrogenesis in vitro
In addition to the involvement of chondrocytes in natural body process, manipulation of chondrocytic precursors in vitro is becoming increasingly important for xe2x80x9ctissue engineeringxe2x80x9d methodologies.
For example, a population of MPCs can be manipulated in vitro such that a majority of cells become chondrocytes (see U.S. Pat. No. 5,908,784 by Johnstone et al.). One use of such systems is to correct and repair cartilage defects through implantation into humans of such chondrocytes derived from differentiation of MPCs in vitro (for example, see U.S. Pat. No. 6,242,247 by Rieser, et al.).
Because tissue-engineered cartilage is a possible treatment for cartilaginous defects, there is a need for probes and detection methods to ensure that mesenchymal cells have differentiated into chondrocytes during the in vitro production of the cartilage.
Differentiation of MPCs as Related to Cancer
Chondrosarcoma is the second most common form of bone malignancy. These are generally slow growing sarcomas that are of unknown etiology and the cell type that initiates the formation of a chondrosarcoma within a bone is not known. Such cells, however, are characterized by the production of cartilage within the sarcoma by cells that differentiate into chondrocyte-like cells. In a specific type of chondrosarcoma, called xe2x80x9cmesenchymal chondrosarcoma,xe2x80x9d cells of the tumor comprised all differentiation stages between and including MPCs and hypertrophic chondrocytes (Aigner, et al., 2000, Am J Pathol, 156:1327-35.).
Conventional chondrosarcoma tumors are graded from stage I through stage III, stage III being the most advanced. Such grading of chondrosarcomas is important for proper diagnosis and treatment of the condition. However, diagnosis and grading of chondrosarcoma has been problematic. For example, the criteria used to distinguish benign enchondroma from low grade chondrosarcoma include parameters which are difficult to quantify such as increased cellularity and more than occasional binucleate cells. These histologic criteria are not absolute, and the diagnosis is frequently made by taking into account clinical features such as pain, rate of growth, location, and radiologic features.
Because it is difficult to stage these sarcomas, there is a need for probes and better detection methods to aid in the definition of the grade of a given chondrosarcoma to assist in the decision-making process for treatment.
The present invention provides new markers which can be used for detecting and staging chondrogenesis in cells. In one aspect, the markers are isolated polynucleotides, referred to hereinafter as CZF-1 and CZF-2, and fragments thereof. In one embodiment, the CZF-1 polynucleotide comprises the sequence set forth in SEQ ID NO. 1. In one embodiment, the CZF-2 polynucleotide comprises the sequence set forth in SEQ ID NO. 3. In another aspect the markers are antibodies which are immunospecific for the proteins encoded by CZF-1 or CZF-2.
The present invention also provides methods which employ the present markers to identify cells that have begun to differentiate into chondrocytes. Such cells are referred to hereinafter as xe2x80x9ccells of interestxe2x80x9d. In one aspect, the method involves contacting the CZF-1 polynucleotide or a fragment thereof, or the CZF-2 polynucleotide or a fragment thereof, with RNA that has been extracted from or, alternatively, contained within the cells of interest, and assaying for the presence of a hybridization product between the polynucleotide and the RNA. In another aspect, the RNA is reverse transcribed and amplified using primers that have been derived from the CZF-1 or CZF-2 polynucleotides. In a further aspect, the method comprises contacting the cells of interest with antibodies that are immunospecific for the CZF-1 protein or the CZF-2 protein and assaying for the formation of an antigen-antibody complex between the anti-CZF-1 or anti-CZF-2 antibody and a protein in the cell.
The present invention also relates to the CZF-1 protein and the CZF-2 protein. Such proteins are useful for preparing antibodies that are used in the present methods for characterizing cells. The present invention also relates to polynucleotides or oligonucleotides whose sequences are complementary to the coding sequences of CZF-1 or CZF-2, or regions thereof. Such polynucleotides and oligonucleotides are useful as probes or primers.